Tumors create local suppression in the tumor microenvironment. They also create widespread systemic unresponsiveness to their own antigens. How this local suppression is transformed into systemic inhibition is not known. We now show that two potent immunosuppressive mechanisms - indoleamine 2,3-dioxygenase (IDO), and the PD-1/PD-ligand pathway - function together as a single linked system to activate regulatory T cells (Tregs) in the tumor microenvironment, and then maintain this activation as Tregs migrate systemically to draining lymph nodes and sites of inflammation. At the molecular level, we show that both IDO and PD-1 signaling converge on the Akt->mTOR pathway in Tregs. During beneficial inflammation, Akt->mTOR signaling normally acts to suspend (de-activate) local Treg suppressor activity, by driving rapid downregulation of the transcription factor FoxO3a in Tregs, thus preventing up-regulation of PD-1. However, when IDO is present, this critical de-activation pathway is blocked, because the Akt->mTOR pathway in Tregs is directly inhibited by IDO (via GCN2 kinase). The inhibition of Akt->mTOR (and thus the expression of FoxO3a and PD-1) are then maintained in an autocrine fashion by signaling through PD-1 on the Tregs (via PTEN phosphatase), independent of IDO. When these IDO-activated, PD-1+ Tregs reach their new sites, they rapidly convert the local DCs into a tolerogenic phenotype characterized by high expression of PD-L1 and PD-L2, low co-stimulation, and defective antigen presentation. Thus, local expression of IDO in the tumor milieu acts as an upstream driver of widespread systemic PD-ligand expression. Aim 1 will use mice with targeted Treg-specific gene deletions to test the mechanistic role of the IL-6-receptor, GCN2 kinase, PTEN phosphatase and FoxO3a transcription factor in Tregs, as mediators of IDO-induced local and systemic suppression. Aim 2 will test the hypothesis that IDO-activated, PD-1+ Tregs create downstream suppression by converting new target DCs into a tolerogenic phenotype with high PD-Ligand expression, via a pathway dependent on reciprocal activation of the FoxO3a transcription factor in DCs. Aim 3 will use translationally-relevant preclinical models of vaccination and chemotherapy to test the hypothesis that simultaneous blockade of the IDO and PD-1 pathways will abrogate excessive Treg activation, both at sites of vaccination and locally within the tumor milieu, and thus allow robust cross-presentation of tumor antigens. Overall, the long-term goal of the current proposal is to abrogate suppression by the potently suppressive population of IDO-activated Tregs, by targeting the linked IDO/PD-1 Treg activation pathway in tumor-bearing hosts.